The complete chloroplast genome sequence of Ficus pandurata Hance var. angustifolia W.C. Cheng (Moraceae)

Abstract Ficus pandurata var. angustifolia is an edible plant popular throughout China where it has a long history of use in traditional She medicine. Using Illumina sequencing, we assembled and annotated the complete chloroplast (cp) genome of F. pandurata var. angustifolia which is 160,526 bp and encodes 130 genes, comprised of 85 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. Phylogenetic analysis resolved F. pandurata var. angustifolia as sister to F. deltoidea which together formed a clade with F. heteromorpha. This complete cp genome sequences is a valuable resource for future studies of evolution and species delimitation in genus Ficus as well as variety breeding and conservation for this species.

Ficus pandurata Hance var. angustifolia W.C. Cheng 1934 is a shrub known as Xiaoxianggou in the traditional She medicine of eastern and southeastern China (Nie et al. 2016) where it is used to treat infantile malnutrition, indigestion, diarrhea, hernia, gouty arthritis, arthralgias, and other diseases (Ying et al. 2012;Wang et al. 2015;Fan et al. 2016). Many Ficus species are used for food and medicine and the sweet fragrance dried F. pandurata var. angustifolia material lends itself to culinary use. Therefore, F. pandurata var. angustifolia is widely cultivated in Zhejiang Province and has received New Resource Food Certification by the local government.
However, there are virtually no genomic resources for the study and conservation of this important species. Here, we describe the first sequenced and annotated chloroplast (cp) genome for F. pandurata var. angustifolia.
Fresh leaves of F. pandurata var. angustifolia were collected from Lishui University (28 27 0 N, 119 54 0 E) in August 2021. The specimen was deposited in Shanghai Chenshan Botanical Garden (Shanghai, China) (Binjie Ge, gebinjie@ csnbgsh.cn) under accession number CSH0192340. This study was approved by the Ethics Committee of Lishui University. Total genomic DNA was extracted using the E.Z.N.A. V R plant DNA kit (Omega Bio-tek, Inc., Norcross, GA), the quality and integrity of extracted DNA were assessed by agarose gel electrophoresis and NanoDrop 2000 (Thermo, Inc., Waltham, MA) spectrophotometry. A DNA library with a 300-500 bp insert was constructed using the Truseq TM DNA sample Prep Kit (Illumina, Inc., San Diego, CA). Next-generation sequencing was conducted by Shanghai Origingene Bio-pharm Technology Co. Ltd. (Shanghai, China) with Illumina NovaSeq6000 platform (Illumina, Inc., San Diego, CA). About 22.65 G raw data were obtained after sequencing and filtered using FastQC (version 0.11.4) and Cutadapt (version 1.16) (Brown et al. 2017). We used the software Fast-plast (version 1.2.8) (https://github.com/mrmckain) and Geseq to assemble and annotate the cp genome (Tillich et al. 2017).
The cp genome of F. pandurata var. angustifolia was 160,526 bp in length, containing a large single-copy region (LSC) of 88,660 bp, a small single-copy region (SSC) of 20,102 bp, and two inverted repeat regions (IRs) of 25,882 bp. The overall GC content is 35.89%, and the values of the LSC, SSC, and IRs regions are 33.52%, 28.97%, and 42.62%, respectively. The genome contains 130 genes including 85 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. These values were similar to the cp genomes of other species from genus Ficus (Chen et al. 2020;Wang and Cui 2020;Xu et al. 2021).
In order to determine the phylogenetic position of F. pandurata var. angustifolia, 38 species with five outgroup taxa (Broussonetia kurzii, Trophis scandens, Morus notabilis, Morus cathayana, and Antiaris toxicaria) were used for phylogenetic analysis. Genome sequences were aligned by using MAFFT (version 7.158b) (Katoh and Standley 2013), and a maximumlikelihood phylogenetic tree was constructed by using RAxML (version 8.2.12) with 1000 bootstrap replicates (Stamatakis 2014). The molecular phylogeny revealed that the systematic position of F. pandurata var. angustifolia as sister to F. deltoidea which together formed a clade with F. heteromorpha (Figure 1). In a previous study using cp intergenic spacer data, these two species came out in a monophyletic assemblage of other species of subgenus Ficus, section Ficus, subsection Frutescentiae (Li et al. 2012). However, its autonym variety Ficus pandurata is found in a well-supported sister relationship to Ficus formosana (Figure 1), which was consistent with a recent report (Huang et al. 2022). In concordance with morphological differences between F. pandurata var. angustifolia and its autonym variety, this complete cp genome sequence of F. pandurata var. angustifolia provides insight into the genetic underpinnings of subspecific diversification in subsection Frutescentiae. This study provides the basis for future phylogenomic studies of F. pandurata var. angustifolia and the phylogenetic relationships within genus Ficus.

Author contributions
Xiaoqing Zhang: conceptualization, methodology, writing original draft, project administration, and writing review and editing; Fei Xu: validation, resources preparing, and visualization; Ling Guo: investigation and formal analysis. All authors agree to be accountable for all aspects of the work and have approved this version to be published.

Disclosure statement
No potential conflict of interest was reported by the authors.

Data availability statement
The genome sequence data generated in this study are openly available in GenBank of NCBI (https://www.ncbi.nlm.nih.gov/) under the accession no. OL639015. The associated BioProject, BioSample, and SRA numbers are PRJNA783434, SAMN23429730, and SRR17035630, respectively.


s2fieldsofstudy: Biology Physics

text:
Nonequivalent After-Effects of Alternating Current Stimulation on Motor Cortex Oscillation and Inhibition: Simulation and Experimental Study

The effects of transcranial alternating current stimulation (tACS) frequency on brain oscillations and cortical excitability are still controversial. Therefore, this study investigated how different tACS frequencies differentially modulate cortical oscillation and inhibition. To do so, we first determined the optimal positioning of tACS electrodes through an electric field simulation constructed from magnetic resonance images. Seven electrode configurations were tested on the electric field of the precentral gyrus (hand motor area). We determined that the Cz-CP1 configuration was optimal, as it resulted in higher electric field values and minimized the intra-individual differences in the electric field. Therefore, tACS was delivered to the hand motor area through this arrangement at a fixed frequency of 10 Hz (alpha-tACS) or 20 Hz (beta-tACS) with a peak-to-peak amplitude of 0.6 mA for 20 min. We found that alpha- and beta-tACS resulted in larger alpha and beta oscillations, respectively, compared with the oscillations observed after sham-tACS. In addition, alpha- and beta-tACS decreased the amplitudes of conditioned motor evoked potentials and increased alpha and beta activity, respectively. Correspondingly, alpha- and beta-tACSs enhanced cortical inhibition. These results show that tACS frequency differentially affects motor cortex oscillation and inhibition.

Recently, transcranial alternating current stimulation (tACS), which involves the noninvasive delivery of a weak alternating current to the scalp, has been used to modulate cortical oscillatory activity and excitability in a frequency-specific manner [9][10][11][12][13]. Such modulatory effects have been reported to occur not only during but also after stimulation [9][10][11]. A possible mechanism underlying tACS-induced after-effects is spike-timing-dependent plasticity (STDP) [14]. In STDP, the pre-and post-synaptic potentials resulting from the rhythm of electrical stimulation-derived neuronal excitation, together with the intrinsic oscillatory patterns of the potentials themselves, affect the magnitude and direction of synaptic strength, leading to synaptic long-term potentiation (LTP) or long-term depression (LTD) [10,14,15]. Previous studies have not only shown that alpha-tACS increased the alpha-band power of brain oscillations but also have shown that this effect outlasted the end of stimulation by ≥30 min [10][11][12] and that the strength of the entrained oscillatory power was positively correlated with the strength of the after-effects [16,17]. These results imply that the effects of tACS on brain oscillation and excitability may last beyond stimulation.
Several studies have suggested that alpha and beta oscillations are negatively correlated with CE [18,19], and others have reported decreased corticospinal excitability after 15 Hz and 20 Hz tACS [19,20]. However, some studies have reported no such changes in excitability after 10 Hz and 20 Hz tACS [19,21,22], while one study reported increased corticospinal excitability after 20 Hz tACS [23]. Therefore, the after-effects of tACS remain controversial. One of the reasons for these inconsistencies is the inter-individual variability in electric fields, which is attributable to the different brain anatomy of each individual [24]. Electric fields are unevenly distributed on the cortex, and little is known about the optimal electrode sites and configurations for successful tACS. Accordingly, although it is known that the timing between electrical stimulation and intrinsic neuronal oscillation affects synaptic strength, the effects of frequency on cortical excitability during and after tACS are not fully understood.
To elucidate this topic, computational modeling of the head of each participant can be used to guide the optimal placement of electrodes [25][26][27], which could also aid in predicting the effects of stimulation [28]. Regardless, this method has limited application in the clinical setting in that it requires imaging data for each participant with neurological and mental disorders such as Alzheimer's disease and Parkinson's disease [29][30][31][32][33]. One promising approach to solve this is to determine the optimal electrode location for a group of participants based on the montage arrangement that delivers the highest intensity with the lowest individual variability [34,35]. The detection of optimal electrode location with highest intensity and lowest variability could be useful for clinical application by mitigating the need for individual imaging data. Therefore, the present study aimed to explore whether optimal tACS electrode montage arrangement could be obtained from individualized head models analyzed at the group level and to examine the effects of alpha-and beta-tACS delivered through such arrangement on cortical excitability. We hypothesized that, if tACS results in a frequency-specific, STDP-mediated strengthening or weakening of neuronal circuits [14,17], then alpha-and beta-band oscillations would change according to tACS frequency. In particular, 10 Hz oscillations (i.e., alpha-band) would be synchronized with the peak phase of 10 Hz and 20 Hz tACSs ( Figure 1A). Similarly, 20 Hz oscillations (i.e., beta-band) would be synchronized with the peak phase of 20 Hz tACS, as well as both the peak and trough phases of 10 Hz tACS ( Figure 1B). Correspondingly, the magnitude of cortical inhibition would change with the increasing power of cortical oscillation resulting from different tACS frequencies. Exploring how cortical oscillations and inhibition change after alpha-and beta-tACS may contribute to our understanding of tACS-induced organizational processes.  neural firing. We hypothesized that (A) 10 Hz oscillations would be synchronized with the peak phase of 10 Hz and 20 Hz tACS, and (B) 20 Hz oscillations would be synchronized with both the peak and trough phases of 10 Hz tACS, as well as the peak phase of 20 Hz tACS.

Participants
Our experimental procedures were approved by the Research Ethics Committee of the Tokyo Kasei University (SKE2018-6) and followed the principles of the Declaration of Helsinki. All participants provided written informed consent prior to participation. In addition, all experiments were performed following the "Guidelines for TMS/tES clinical services and research through the COVID-19 pandemic" [36].
This was a single-center, single-blinded, within-participant study. The selection of the sample size was based on a desired statistical power of 80% for the detection of changes in power spectra and motor evoked potential (MEP) amplitudes, with an effect size of 0.30 and a two-sided α-level of 0.05. According to these parameters, G*Power 3.0 [37] yielded a sample size of 16. Therefore, we recruited 16 healthy volunteers without neurological or psychiatric diseases who were not at risk of adverse events from transcranial magnetic stimulation (TMS) [38] and were not taking any medication. Right-handedness was confirmed with the Edinburgh Handedness Inventory [39].

Electric Field Simulation
A volume conductor model of the anatomical human head model was constructed using magnetic resonance imaging (MRI) images from a database of eighteen participants (all healthy males). The generated tACS electric field was conducted for the following scenarios. We considered seven tACS montages based on the International 10-20 system and anatomical structure ( Figure 2A). tACS was applied with an intensity of 0.3 mA throughout the rubber sheets (1.8 × 1.8 cm 2 ) corresponding to a stimulation phase. We compared the normal component of the electric field values averaged over the precentral knob in the precentral area ( Figure 2B) in the standard brain space among all electrode configurations for group-level analysis. Additionally, relative standard deviation (RSD) was used to quantify how much variability of the electric field was present between participants. Supplementary data presents the detailed computational model implementation.

Hotspot Detection
Each participant was comfortably seated with their right hand resting on the testing equipment. The skin overlying the right first dorsal interosseous (FDI) muscle was cleaned with alcohol to reduce its electrical resistance, and recording and reference double differential surface electrodes (FAD-DEMG1, 4Assist, Tokyo, Japan) were placed over the muscle. MEPs from the FDI muscle were recorded, amplified by 100, bandpass-filtered at  Hz, digitized at 10 kHz with a PowerLab system (ADInstruments, Dunedin, New

Hotspot Detection
Each participant was comfortably seated with their right hand resting on the testing equipment. The skin overlying the right first dorsal interosseous (FDI) muscle was cleaned with alcohol to reduce its electrical resistance, and recording and reference double differential surface electrodes (FAD-DEMG1, 4Assist, Tokyo, Japan) were placed over the muscle. MEPs from the FDI muscle were recorded, amplified by 100, bandpass-filtered at  Hz, digitized at 10 kHz with a PowerLab system (ADInstruments, Dunedin, New Zealand), and stored in a solid-state drive.
After placing a tight-fitting cap over the participant's head, we drew intersecting nasion-inion and interaural lines on the cap with a marker pencil to localize the vertex (Cz) in accordance with the 10-20 International System. Magstim 200 2 (Magstim, Whitland, UK) stimulators were employed to deliver TMS as a monophasic current waveform via a cable to the scalp surface through a figure-of-eight coil (internal diameter of each wing: 70 mm). To induce current flow in the left brain along the posterior-lateral to anterior-medial direction, we placed the coil tangentially to the scalp and held the handle so that it would point backwards and sideways, at approximately 45 • from the midline. As previously described [40,41], we visually detected the optimal coil position to elicit maximum MEPs in the right FDI muscle ("hotspot") and marked the location with a soft-tipped pen.

tACS
To determine the after-effects of tACS frequency on brain oscillations and cortical excitability, each participant was tested with two active (alpha-and beta-tACS) and one sham condition ( Figure 3A) on three different days. sensations such as tingling [43]; no current was delivered for the remaining 19 min and 30 s. The order of conditions was randomized across participants, and all sessions were separated by ≥1 day. Participants were blinded to the condition.

Electroencephalography
Electroencephalographic (EEG) data were obtained before and after tACS. The skin was prepared with alcohol, and five gold-coated active EEG electrodes were placed at the FDI muscle hotspot (HS), and 2.5 cm lateral anterior (LA), medial anterior (MA), lateral posterior (LP), and medial posterior (MP) to the hotspot, respectively ( Figure 3B). Electrodes were mounted in an elastic cap by a holder and covered by support bandages. Electrodes were also placed above the right eye and below the left eye to record activities related to eye movements and blinking.
Participants were instructed to keep their eyes open and fixate on a 0.5 cm blue dot on a screen located about 100 cm in front of them. EEG was performed using the Polymate V (Miyuki Giken, Tokyo, Japan), and data were sampled at 1000 Hz and filtered from 0.15 Hz to 200 Hz. Electrode impedance was maintained at ≤10 kΩ. EEG signals were referenced to the averaged recordings of the right and left earlobes. For all procedures, the participants were seated in a comfortable chair with their eyes open in a quiet room. tACS was delivered by a battery-driven current stimulator (DC Stimulator-Plus; NeuroConn, Ilmenau, Germany) through two rubber electrodes (1.8 × 1.8 cm) attached to the participants' scalp. Using a conductive and adhesive paste (Ten20 Conductive Paste; Weaver and Company, Aurora, CO, USA) and a support bandage, the electrodes were placed above Cz and CP1, respectively, in accordance with the 10-20 International System. The Cz-CP1 montage was selected because, in our simulation, this configuration produced high current densities with low variability in the hand motor area (see Section 3). For active stimulation, tACS was delivered at a fixed frequency of 10 Hz (alpha-tACS) or 20 Hz (beta-tACS) with a peak-to-peak amplitude of 0.6 mA (current density: 0.093 mA/cm 2 ) through a current stimulator for 20 min [22,42]. Sham stimulation was performed at a fixed frequency of 15 Hz with the same intensity for 30 s to cause skin sensations such as tingling [43]; no current was delivered for the remaining 19 min and Brain Sci. 2022, 12,195 5 of 15 30 s. The order of conditions was randomized across participants, and all sessions were separated by ≥1 day. Participants were blinded to the condition.

Electroencephalography
Electroencephalographic (EEG) data were obtained before and after tACS. The skin was prepared with alcohol, and five gold-coated active EEG electrodes were placed at the FDI muscle hotspot (HS), and 2.5 cm lateral anterior (LA), medial anterior (MA), lateral posterior (LP), and medial posterior (MP) to the hotspot, respectively ( Figure 3B). Electrodes were mounted in an elastic cap by a holder and covered by support bandages. Electrodes were also placed above the right eye and below the left eye to record activities related to eye movements and blinking.
Participants were instructed to keep their eyes open and fixate on a 0.5 cm blue dot on a screen located about 100 cm in front of them. EEG was performed using the Polymate V (Miyuki Giken, Tokyo, Japan), and data were sampled at 1000 Hz and filtered from 0.15 Hz to 200 Hz. Electrode impedance was maintained at ≤10 kΩ. EEG signals were referenced to the averaged recordings of the right and left earlobes.

Cortical Inhibition Recordings
To measure cortical inhibition and evaluate GABA A -mediated inhibitory effects, MEPs and short-interval intra-cortical inhibition (SICI) were measured before and after tACS [44,45]. The hotspot's resting motor threshold (RMT) was defined as the minimum stimulus intensity required to elicit an MEP ≥50 µV in the relaxed FDI muscle in 5 out of 10 consecutive trials. Unconditioned MEPs for the FDI muscle were evoked at the hotspot at 120% of the RMT value. The stimulus intensity for the first conditioning pulse was set at 80% of the RMT value, and the second test pulse was administered suprathreshold at an intensity of 120% that of the RMT. A 2.5 ms interstimulus interval was used to test SICI [44,45]. Twenty trials of both unconditioned MEP and SICI measurements at a frequency of 0.2 Hz were recorded in random order.

EEG Data Processing
The six EEG datasets (i.e., data collected before and after alpha-, beta-, and sham-tACS) were each split into 180 non-overlapping 1 s epochs. All epochs were visually inspected, and those containing eye blinks or muscle movement artifacts were excluded. After artifact rejection, the fast Fourier transform was applied for frequencies between 0 and 40 Hz (1 Hz resolution) for individual epochs using a Hanning window. After logarithmically transforming and averaging the power values of the five electrodes, frequency bands of interest were selected in the alpha (10 ± 1 Hz) and beta (20 ± 1 Hz) ranges, taking into account the tACS frequencies.
In order to conduct a proper comparison for differences in power spectra between tACS frequency conditions, normality testing using the Kolmogorov-Smirnov test was used. Based on the result of the Kolmogorov-Smirnov testing, either parametric two-way repeated measures analysis of variance (ANOVA) or nonparametric Friedman's test was used. Additionally, for nonparametric testing, the logarithmically transformed power spectrum without normality distribution was normalized to baseline (i.e., before tACS) according to the following equation: where NP denotes the normalized power spectrum, A represents the EEG power spectrum at time t and frequency f (i.e., the power spectrum of 10 and 20 Hz after tACS), and R denotes the mean power spectrum of the baseline period, defined as the 3 min interval before tACS. A large positive value indicates a large increase in the EEG power spectrum from the baseline period [46]. Furthermore, post hoc analysis with parametric Bonferroni correction or the nonparametric Steel-Dwass test was performed to compare differences in power spectra between tACS frequency conditions.

MEP Data Processing
A previous study [47] noted that MEP amplitudes randomly fluctuate between stimuli. Therefore, peak-to-peak MEP amplitudes were evaluated for the existence of outliers through Tukey's fences, with values more than 1.5 times that of the interquartile range excluded from the datasets [48]. To increase the precision of level and slope estimations of cortical inhibition, the blank cells produced by removing the outliers were then linearly interpolated. Next, time-series analyses were conducted using the Bayesian method. The local linear trend model (LLT) assumes that both the level (Equation (3)) and slope (Equation (4)) of the trend from observational values (Equation (2)) follow Gaussian random walks. LLTs were constructed for the MEP amplitudes as follows: where y t is the observational value; ε t indicates random variables; µ 1, represents the initial level; ν 1 is the initial slope; and ξ t and ζ t indicate disturbances in the level and slope, respectively [49]. After state value estimation, normality testing using Kolmogorov-Smirnov test was used. Based on the result of the Kolmogorov-Smirnov testing, either parametric two-way repeated measures ANOVA or nonparametric Friedman's test was used. For nonparametric testing, conditioned and unconditioned MEP amplitudes were normalized to the baseline data (Equation (1)). In Equation (1), NP denotes normalized MEPs, A denotes MEPs at time t, and R denotes the mean MEP of the baseline period before tACS. A great positive value indicates a large increase in MEPs compared with that in the baseline period [46]. Post hoc analysis with parametric Bonferroni correction or the nonparametric Steel-Dwass test was performed to compare differences in MEP amplitudes among the three tACS conditions. Data analysis was conducted with EMSE (Miyuki Giken, Tokyo, Japan), the SciPy package in the Python environment (Python Software Foundation, Wilmington, DE, USA), and the R 3.4.0 software (The R Foundation, Vienna, Austria). Data are expressed as means ± standard errors of the mean (SEM). Statistical significance was set at p < 0.05. Figure 4 shows the group-level electric field distribution (normal component) on standard cortical brain space for different montages. The electric field was induced in the precentral gyrus for tACS with a 3.24 cm 2 rubber sheet and an intensity of 0.3 mA on each phase. However, the field focality was not identical between the montages. In addition, we compared the averaged electric field values in the hand knob among the montages (Table 1 and Figure 4). The higher averaged values corresponded to Cz-CP1 as 0.12 V/m (min = 0.04 V/m, max = 0.22 V/m). Additionally, we found less variability in the induced electric field among participants for the Cz-CP1 (±standard deviation (SD) = 0.05 V/m, relative SD = 38%) and FC1-Pz (±SD = 0.04 V/m, relative SD = 40%), as shown in Table 1 and Figure 4. In summary, the mean of the normal component averaged over the hand knob was higher and more stable by selecting the Cz-CP1 montage at group-level analysis, and at the same time the Cz-CP1 montage of tACS not interfering with the EEG recording for the M1 region.

Electric Felds of Cortical tACS
(min = 0.04 V/m, max = 0.22 V/m). Additionally, we found less variability in the induced electric field among participants for the Cz-CP1 (± standard deviation (SD) = 0.05 V/m, relative SD = 38%) and FC1-Pz (± SD = 0.04 V/m, relative SD = 40%), as shown in Table 1 and Figure 4. In summary, the mean of the normal component averaged over the hand knob was higher and more stable by selecting the Cz-CP1 montage at group-level analysis, and at the same time the Cz-CP1 montage of tACS not interfering with the EEG recording for the M1 region.

Changes in Brain Oscillation and Excitation
A total of 4 men and 12 women aged 20-40 years (25.1 ± 7.5 years) were enrolled, and the mean laterality quotient score was 0.9 (SD = 0.1). Figure 5 shows the power spectrum grand-averaged across all participants. As shown in Figure 5, the power spectrum of alphaband oscillations was increased after alpha-tACS, whereas that of beta-band oscillations was increased after beta-tACS. However, sham-tACS did not result in any changes in either power spectrum.
The Kolmogorov-Smirnov test showed that the power spectra lacked normality (alpha oscillation before and after alpha-tACS, beta-tACS, and sham-tACS: both p < 0.0001). Therefore, nonparametric testing and Equation (1) was used for comparison of the power spectra of alpha and beta oscillations after alpha-, beta-, and sham-tACS treatments. The normalized power changes (i.e., the event-related synchronization (ERS) and event-related desynchronization (ERD)) in alpha-band oscillatory neural activities after alpha-, beta-, and sham-tACSs are shown in Figure 6A. The Friedman test showed a significant difference in power changes in alpha-band oscillations among alpha-, beta-, and sham-tACSs (chisquared = 16.75, degree of freedom = 2, p = 0.0002). Additionally, post hoc tests showed that alpha power oscillation was greater after alpha-tACS than after sham-tACS (alpha-tACS vs. beta-tACS: t = 1.37, p = 0.358; alpha-tACS vs. sham-tACS: t = 3.51, p = 0.001; beta-tACS vs. sham-tACS: t = 2.29, p = 0.057). Alpha power oscillation was greater after beta-tACS than after sham-tACS, but significance was not reached. A total of 4 men and 12 women aged 20-40 years (25.1 ± 7.5 years) were enrolled, and the mean laterality quotient score was 0.9 (SD = 0.1). Figure 5 shows the power spectrum grand-averaged across all participants. As shown in Figure 5, the power spectrum of alpha-band oscillations was increased after alpha-tACS, whereas that of beta-band oscillations was increased after beta-tACS. However, sham-tACS did not result in any changes in either power spectrum. Dashed and solid lines denote the power spectra before and after tACS, respectively. Shaded areas indicate the standard error of the mean. The power spectrum of alpha-band oscillation was increased after alpha-tACS, whereas beta-band oscillation was increased after beta-tACS. However, the power spectra of alpha-and beta-band oscillations were not changed by sham-tACS.
The Kolmogorov-Smirnov test showed that the power spectra lacked normality (alpha oscillation before and after alpha-tACS, beta-tACS, and sham-tACS: both p < 0.0001). Therefore, nonparametric testing and Equation (1) was used for comparison of the power spectra of alpha and beta oscillations after alpha-, beta-, and sham-tACS treatments. The normalized power changes (i.e., the event-related synchronization (ERS) and event-related desynchronization (ERD)) in alpha-band oscillatory neural activities after alpha-, beta-, and sham-tACSs are shown in Figure 6A. The Friedman test showed a significant difference in power changes in alpha-band oscillations among alpha-, beta-, and sham-tACSs (chi-squared = 16.75, degree of freedom = 2, p = 0.0002). Additionally, post hoc tests showed that alpha power oscillation was greater after alpha-tACS than after sham-tACS (alpha-tACS vs. beta-tACS: t = 1.37, p = 0.358; alpha-tACS vs. sham-tACS: t = 3.51, p = 0.001; beta-tACS vs. sham-tACS: t = 2.29, p = 0.057). Alpha power oscillation was greater after beta-tACS than after sham-tACS, but significance was not reached. Dashed and solid lines denote the power spectra before and after tACS, respectively. Shaded areas indicate the standard error of the mean. The power spectrum of alpha-band oscillation was increased after alpha-tACS, whereas beta-band oscillation was increased after beta-tACS. However, the power spectra of alpha-and beta-band oscillations were not changed by sham-tACS. The ERS/ERD of beta-oscillatory neural activities after alpha-, beta-, and sham-tACSs are shown in Figure 6B. The Friedman test showed a significant difference in the ERS/ERD of beta oscillations among the alpha-, beta-, and sham-tACSs (chi-squared = 11.53, degree of freedom = 2, p = 0.003). Additionally, post hoc tests showed that beta power oscillation was greater after beta-tACS than after sham-tACS (alpha-tACS vs. beta-tACS: t = 1.65, p = 0.358; alpha-tACS vs. sham-tACS: t = 3.45, p < 0.0001; beta-tACS vs. sham-tACS: t = 4.34, p = 0.016). Moreover, beta power oscillation was lower after alpha-tACS than after sham-tACS.
The grand-averaged actual and estimated peak-to-peak MEP amplitudes according to the LLT are shown in Figure 7. The actual MEP amplitudes fluctuated randomly before and after alpha-, beta-, and sham-tACS, whereas the fluctuation in the estimated MEP amplitudes was reduced by the LLT.
The Kolmogorov-Smirnov test showed that the MEP amplitudes lacked normality (conditioned and unconditioned MEP before and after alpha-tACS, beta-tACS, and sham-tACS: both p < 0.0001). Therefore, nonparametric testing and Equation (1) was used for comparison of the conditioned and unconditioned MEP amplitudes after alpha-, beta-, and sham-tACS treatments. The changes in the normalized conditioned MEP amplitudes after tACS are shown in Figure 8A. The Friedman test showed a significant difference in normalized condition MEP amplitudes among alpha-, beta-, and sham-tACSs (chisquared = 42.28, degree of freedom = 2, p < 0.0001). Further, post hoc tests showed that the The ERS/ERD of beta-oscillatory neural activities after alpha-, beta-, and sham-tACSs are shown in Figure 6B. The Friedman test showed a significant difference in the ERS/ERD of beta oscillations among the alpha-, beta-, and sham-tACSs (chi-squared = 11.53, degree of freedom = 2, p = 0.003). Additionally, post hoc tests showed that beta power oscillation was greater after beta-tACS than after sham-tACS (alpha-tACS vs. beta-tACS: t = 1.65, p = 0.358; alpha-tACS vs. sham-tACS: t = 3.45, p < 0.0001; beta-tACS vs. sham-tACS: t = 4.34, p = 0.016). Moreover, beta power oscillation was lower after alpha-tACS than after sham-tACS.
The grand-averaged actual and estimated peak-to-peak MEP amplitudes according to the LLT are shown in Figure 7. The actual MEP amplitudes fluctuated randomly before and after alpha-, beta-, and sham-tACS, whereas the fluctuation in the estimated MEP amplitudes was reduced by the LLT.   The Kolmogorov-Smirnov test showed that the MEP amplitudes lacked normality (conditioned and unconditioned MEP before and after alpha-tACS, beta-tACS, and sham-tACS: both p < 0.0001). Therefore, nonparametric testing and Equation (1) was used for comparison of the conditioned and unconditioned MEP amplitudes after alpha-, beta-, and sham-tACS treatments. The changes in the normalized conditioned MEP amplitudes after tACS are shown in Figure 8A. The Friedman test showed a significant difference in normalized condition MEP amplitudes among alpha-, beta-, and sham-tACSs (chisquared = 42.28, degree of freedom = 2, p < 0.0001). Further, post hoc tests showed that the conditioned MEP amplitudes were smaller after alpha-and beta-tACS than after sham-tACS. Specifically, MEP amplitudes after alpha-tACS were smaller than those after beta-tACS (alpha-tACS vs. beta-tACS: t = 2.56, p = 0.029; alpha-tACS vs. sham-tACS: t = 4.93, p < 0.0001; beta-tACS vs. sham-tACS: t = 2.38, p = 0.045).

Discussion
Previous studies did not conduct a group-level evaluation of the optimal sites and montage configurations for tACS electrodes [7,13,[19][20][21][22][23]42]. Therefore, we investigated this through a computational simulation, which showed that the tACS Cz-CP1 montage arrangement diminishes the inter-individual variability in the electric field. The electric field range for this arrangement (0.1 V/m-0.2 V/m) indicated a modulatory effect [50]. Therefore, we utilized the Cz-CP1 montage configuration to deliver tACS. We opted for a group-level electric field analysis to maximize the electric field on the target while minimizing individual variability to determine the optimal montage applied to all participants (one-for-all) based on the International 10-20 system positioning. This is advantageous with respect to the individual-level electric field analysis, as it does not require imaging of each individual or electrode localization based on a navigation system that is not always available in clinical settings, and it did not increase the participants' time in the experiment, which was limited during the COVID-19 pandemic [36]. Therefore, our simulation based on group-level electric field analysis is advantageous for adaptation to various clinical settings, obviating the need for imaging data in the individual-level electric field analysis.
Our experimental results show that alpha-and beta-tACS result in larger alpha and beta oscillations, respectively, and they differently influence cortical inhibition. In addition, alpha-, beta-, and sham-tACS result in a stepwise decrease in conditioned MEP amplitudes. These observations show that alpha-and beta-tACS differently modulate alpha-and betaband oscillations, which, in turn, differently influence cortical inhibition. This implies that cortical oscillation and inhibition are not equally affected by alpha-and beta-tACSs. Previous studies have noted that tACS has online and offline modulatory effects during and after stimulation [9][10][11]. Especially, the offline effects after tACS underline its potential as a therapeutic tool because of its lasting effect beyond stimulation period.
Two possible mechanisms of tACS modulatory effect have been suggested. First, tACS directly entrains intrinsic brain oscillations [51][52][53][54]. Second, tACS leads to synaptic changes via STDP mechanisms [10,14,15,17]. In entrainment of brain oscillation, intrinsic brain oscillation in accordance with the external stimulation frequency will be entrained but intrinsic brain oscillation outside the stimulation frequency will not be affected [11,55]. Therefore, intrinsic alpha and beta oscillation in M1 [56,57] are externally tuned by tACS, according to the resonance-like hypothesis [23]. Adding to the entrainment mechanism, STDP could possibly explain tACS-induced after-effects [14]. Synapses are either strengthened or weakened depending on the timing of their input and output activity [10,14,15,17], which might be related to the after-effects of tACS [58]. According to the STDP model, facilitatory effects on neural oscillations are expected with the synchronization between the peak phase of tACS and the up state of neural firing, whereas depressive effects are expected with the asynchronization between the two events (i.e., the trough phase of tACS synchronizing the up state of neural firing) [10,14,15,17,55,58]. In agreement with these predictions, we observed that alpha-and beta-tACS increased alpha and beta oscillatory activity, respectively, while alpha-tACS decreased beta oscillatory activity. One possible explanation for this decrement could be that both the peak and trough phases of alpha-tACS may synchronize the up state of neural firing. Previous studies have already showed an enhanced oscillatory activity after tACS [10,14,15,17,58], but we found that tACS frequency influences brain oscillatory activity in a frequency-dependent manner.
Kiers et al. [47] noted that MEP amplitudes randomly fluctuated during the recording period, and Ogata et al. [18] suggested that, during the resting state, the relationship between amplitude fluctuations and cortical oscillations and inhibition is not conclusive. To solve these issues, we used the LLT model based on Bayesian estimations to examine the effects of alpha-and beta-tACS on the level and slope of cortical inhibition, thus eliminating the confounding factor of amplitude fluctuations during the resting state.
Alpha-and beta-band oscillations have been associated with the inhibitory GABAergic interneurons within M1 [4][5][6]59]. Accordingly, enhanced GABAergic interneuron activity plays an essential role in the modulation of M1 excitability induced by alpha-and beta-tACS [60]. However, a previous study found no effect of beta-tACS in SICI after TMSinduced GABA A inhibition [7,45]. Our findings on the alpha-and beta-tACSs effects on SICI suggest that the cortex is inhibited after stimulation. One possible explanation for this discrepancy could be that changes in the M1 post-synaptic potentials that correspond to alpha-and beta-oscillations can be altered by alpha-and beta-tACS, assuming that tACS enhances the synaptic strength of GABAergic neurons, as described by the STDP model [10,14,15,17,58]. Therefore, cortical inhibition might be affected by the enhancement of cortical oscillations associated with tACS frequency.
Previous work has shown that beta oscillations correlate with CE levels [61][62][63] and that alpha and beta oscillations inhibited MEP amplitudes [18,[64][65][66], although a statistically significant relationship has not always been observed [67,68]. In addition, several studies noted that 20 Hz tACS increased corticospinal excitability [13,23]. However, Cappon et al. [69] and Zaghi et al. [20] observed reduced MEP amplitudes after 15 and 20 Hz tACS of M1, and Wach et al. [21] and Schutter and Hortensius [19] did not find any effect of 10 and 20 Hz tACS. However, in our study, unconditioned MEP amplitudes decreased after alpha-and beta-tACSs. Sanger et al. [70] suggested that MEP amplitude was affected not only by GABA A receptors, thus suggesting SICI, but also by excitatory glutamatergic and inhibitory GABA B receptors. However, the precise mechanism of tACS-related changes in unconditioned MEP amplitude is still unclear. Future studies should investigate changes in CE and inhibition related to GABA A , GABA B , and glutamatergic receptors at various tACS frequencies.
Previous studies indicate that neurological and mental diseases induce changes in brain oscillations [29][30][31][32][33]71,72]. In Parkinson's disease, abnormal beta activity could be related to bradykinesia [73]. Additionally, in Alzheimer's disease, abnormal alpha activity could be related to memory dysfunction [74]. The frequency-specific tACS modulatory effects for brain oscillation and inhibition could have potentially useful clinical applications. Further studies are needed to assess the tACS modulatory effect on brain oscillatory and inhibitory disorders in neurological and mental disorders such as Parkinson's disease and Alzheimer's disease.
Our study has two main limitations. First, because it was necessary to monitor tACS waves for safe and precise stimulation, we could not utilize a double-blinded design. Therefore, double-blinded studies should be conducted in the future. Second, the sample size and composition, which was estimated using G*Power 3.0 [24], were limited as we did not consider factors such as differences in age, sex, baseline MEP sizes, or MEP's latency. In fact, concerning these two latter factors, Wiethoff et al. [75] noted that baseline MEP sizes and latency differences in MEP (anteroposterior stimulation minus latero-medial stimulation) result in changes in corticospinal excitability. Thus, future studies need to include a larger sample size to analyze the effects of such parameters on cortical excitability.

Conclusions
In conclusion, we found that changes in tACS frequency result in corresponding changes in alpha-and beta-band oscillations and cortical inhibition. These results imply that cortical oscillations can be differentially altered by tACS and that cortical inhibition may change according to the tACS frequency-modulated balance between alpha and beta oscillations.


Anti-Osteoporotic Effect of Viscozyme-Assisted Polysaccharide Extracts from Portulaca oleracea L. on H2O2-Treated MC3T3-E1 Cells and Zebrafish

This study aims to screen and characterize the protective effect of polysaccharides from Portulaca oleracea L. (POP) against H2O2-stimulated osteoblast apoptosis in vivo and in vitro. The enzymes viscozyme, celluclast, α-amylase, and β-glucanase were used to extract POPs. Among all enzyme-assisted POPs, the first participating fraction of viscozyme extract POP (VPOP1) exhibited the highest antioxidant activity. Hoechst 33342 and acridine orange/ethidium bromide staining and flow cytometry of MC3T3 cells revealed that VPOP1 inhibited apoptosis in a dose-dependent manner. Moreover, VPOP1 increased the expression levels of heme oxygenase-1 (HO-1) and NADPH quinine oxidoreductase 1 (NQO1) and decreased the expression levels of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) in H2O2-induced cells compared with their controls. The results of an in vivo experiment show that VPOP1 significantly reduced reactive oxygen species generation and lipid peroxidation in zebrafish at 72 h post-fertilization and promoted bone growth at 9 days post-fertilization. Furthermore, VPOP1 was identified via 1-phenyl3-methyl-5-pyrazolone derivatization as an acidic heteropolysaccharide comprising mannose and possessing a molecular weight of approximately 7.6 kDa. Collectively, VPOP1 was selected as a potential anti-osteoporotic functional food because of its protective activity against H2O2-induced damage in vitro and in vivo.

Introduction
With the intensification of global aging, osteoporosis (OP) has gradually become an international research hotspot [1]. OP is a systemic metabolic disease characterized by bone mass reduction, bone microstructure destruction, and bone fragility increase [2]. Bone loss is related to a lack of estrogen [3], and age-related OP is closely related to oxidative stress [4]. Bone growth and maintenance require mesenchymal stem cells (MSCs) for osteogenic differentiation [5]. MSCs undergo metabolic switches such as reduced glycolysis and increased mitochondrial respiration to ensure adequate energy supply for differentiation [6]. In cells, mitochondrial electron transport is the main mechanism for reactive oxygen species (ROS) production [7]. Hence, an increase in mitochondrial metabolism is usually accompanied by an increase in endogenous ROS. During normal metabolism, ROS can be scavenged by antioxidants [8]. However, oxidative stress occurs when ROS production is not counterbalanced by the antioxidant activity of cells [9]. Oxidative stress can decrease osteoblast (OB) activity, induce OB apoptosis to accelerate bone flow, and promote osteoclast (OC) differentiation to disrupt bone homeostasis [10]. The current treatment for OP is estrogen therapy, but it has several non-skeletal adverse consequences [11]. Recent studies have strongly recommended the use of extracts from natural products to prevent OP, as the

Polysaccharides Extracted from POL
Four different enzymes, including viscozyme, celluclast, α-amylase, and β-glucanase, were employed for polysaccharide extraction. POL was purchased from a pharmacy in Changchun, Jilin Province, China. POL was sun-dried and ground, then soaked in petroleum ether for 24 h. The defatted powder was suspended in 95% ethanol for 24 h to remove any pigment and small molecules. The ethanol was removed using a vacuum evaporator. Subsequently, the dried powder was mixed with 1% enzyme and suspended in deionized water at a ratio of 1:15 (g mL −1 ). In order to avoid the protein being highly insoluble and hindering the release of biomolecules [24], various enzymes were selected to obtain the optimum pH. The optimum pH of viscozyme and celluclast was 4.5, and that of α-amylase and β-glucanase was 6.0. Therefore, the mixture was divided into four flasks, in which the pH was adjusted to 4.5, 4.5, 6.0, and 6.0, respectively, by diluted HCl and NaOH. Viscozyme-and celluclast-assisted aqueous extraction was agitated for 8 h at 50 • C, whereas α-amylaseand β-glucanase-assisted aqueous extraction was agitated for 8 h at 60 • C. The particles were removed using a large-scale filter. Four enzymes in the supernatants were denatured by incubating the mixture at 100 • C for 20 min. The supernatants were treated with Sevag reagent to remove proteins [25], and then the collected supernatants were regarded as crude POP.

Survival Rate and ROS Detection
Mouse OB cell line MC3T3-E1 was purchased from the American Type Culture Collection. Depending on the different degrees of influence on the cell state, the appropriate concentration was screened and determined as the modeling concentration; POPs with different concentrations were used to intervene in the damaged cells. A colorimetric 3-(4,5)dimethylthiahiazo(-z-y1)-2,5-diphenytetrazoliumromide (MTT; Solarbio, Beijing, China) assay was carried out to detect cell viability in H 2 O 2 -stimulated MC3T3 cells [27]. A density of 1 × 10 4 cells well −1 MC3T3 cells were seeded in 96-well plates. Upon reaching 80% confluence, the cells were added to increasing concentrations (12.5-100 µg mL −1 ) of POPs, and then treated with H 2 O 2 for 24 h. Subsequently, each plate was added to 50 µL of PBS solution with 2 mg mL −1 of MTT. Following incubation for 3 h, the medium was gently removed, and precipitate was dissolved in 200 µL of dimethyl sulfoxide. The absorbance was obtained with a microplate reader at 540 nm. Cell survival rate was calculated as a percentage of formazan absorption in the control cells.
Determination of intracellular ROS levels in MC3T3 cells was performed using a DCFH-DA probe [28]. The cells seeding and sample treating were the same by cell viability assay. After being treated with H 2 O 2 , the cells were treated with 10 µL of DCFH-DA (0.5 mg mL −1 ) after 3 h of incubation. A fluorescence microscope was used to detect the cell fluorescence intensity. Excitation and emission wavelength were operated at 485 nm and 530 nm, respectively.

Hoechst 33342 and Acridine Orange/Ethidium Bromide Staining
Hoechst 33342 and acridine orange/ethidium bromide (AO/EB) staining are morphological probes to reveal apoptotic changes in cells. A density of 1 × 10 5 cells mL −1 MC3T3 cells was pre-seeded in 12-well culture plates, and H 2 O 2 -induced cells were added to increasing concentrations of POPs and incubated for 24 h. Controls were not treated with POPs and H 2 O 2 . For Hoechst 33342 staining, each plate was added to 10 µL of Hoechst 33342 (1 mg mL −1 ) solution for 10 min at room temperature in the dark. For acridine orange/ethidium bromide (AO/EB) staining, 10 µL of AO/EB (100 µg mL −1 ) was added to each well for 10 min at room temperature in the dark, and then washed with PBS three times. The image was then captured under a fluorescence microscope (Nikon, Tokyo, Japan).

Flow Cytometry Analysis
MC3T3 cells were seeded at a density of 2 × 10 5 cells mL −1 in 6-well culture plates and then added to indicated concentrations of POPs. Following a 24 h incubation, the culture medium was rinsed by pre-cooled PBS. The cell pellet was suspended in 1× binding buffer. The cells were continuously stained with PI and Annexin V-FITC (Becton Dickinson, Frankly Lakes, NJ, USA) for 15 min in the dark. Samples were analyzed within 1 h after staining. Cells were analyzed using a flow cytometer system (FlowSight, Merck Millipore, Seattle, WA, USA) and data were analyzed using the IDEAS (FlowSight, Merck Millipore, Seattle, WA, USA).

Western Blot Analysis
A density of 2 × 10 6 cells mL −1 MC3T3 cells was seeded in 6-well culture plates for 24 h incubation. The H 2 O 2 -induced MC3T3 cells were added to increasing concentrations of POPs and then collected after 24 h. The cells were suspended and homogenized in lysis buffer for 1 h. Then, the suspension was centrifuged to remove the pellet. A BCA kit (Solebao, Beijing, China) was employed to detect protein content. Polyacrylamide gels (10%) were loaded with 20 µg of protein from each sample treated with lysis buffer. Different molecular weights of protein were separated by electrophoresis. The separated protein bands were subsequently transferred onto a nitrocellulose membrane. Then, blocking buffer blocked the membrane for 3 h at room temperature. The membrane was incubated with primary antibodies at 4 • C for 8 h, followed by secondary antibodies at room temperature for 3 h. All immunoblots were processed using the Chemiluminescence Substrate Kit (Biosharp, Beijing, China) and visualized through chemiluminescence imaging (Tanon 5200, Shanghai, China). Band intensities were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Analysis of Zebrafish Embryos
AB wild-type zebrafish (Danio rerio) were obtained from EzeRinka Biotechnology Co., Ltd. (Nanjing, China). Zebrafish were incubated in a water tank at 28.5 • C with a light/dark cycle of 14/10 h, and fed three times a day with live brine shrimp (Artemia). Embryos were obtained from natural spawning. The embryos were collected and incubated in E3 medium. The levels of H 2 O 2 -induced ROS and lipid peroxidation in the embryos were evaluated. Each group was randomly set with fifteen embryos into 6-well plates at 7 h post-fertilization (hpf). The embryos were added to concentrations of POPs followed by H 2 O 2 treatment. Fresh medium was added every 24 h until the third day. Fluorescent probes, DCFH-DA and diphenyl-1-pyrenylphosphine, were used to detect ROS production and lipid peroxidation, respectively. The microscopic fluorescence images of the embryos were obtained under a fluorescence microscope (Nikon, Tokyo, Japan).
Each group was randomly set with fifty embryos into Petri dishes at 5 hpf. The embryos were added to H 2 O 2 and POPs at 7 hpf and collected at 72 hpf. The collected embryos were homogenized with lysis buffer, and subsequently for Western blot analysis.
Bone development in the larval stage of zebrafish was visualized using the fluorescent chromophore calcein. The embryos were added to increasing concentrations of POPs and then incubated for 1 h. A fresh medium of H 2 O 2 was added to each well containing 5-day post-fertilization (dpf) embryos. Fresh culture medium was added after every 24 h of incubation and then transferred onto 24-well plates at 9 dpf. After adding calcein-containing solution (2 mg mL −1 ), the embryos were incubated in the dark at room temperature for 5 min and then washed with E3 water until colorless. After zebrafish staining, images of the embryos were obtained under a fluorescence microscope (Nikon, Tokyo, Japan).

Molecular Weight of POPs
High-performance liquid chromatography and gel permeation chromatography (HPLC-GPC) were employed to determine the molecular weight (Mw) of POPs. Dextran standards and POPs were dissolved in 0.7% sodium sulfate solution at a concentration of 10 mg mL −1 and analyzed on an LC-2030C 3D Series HPLC (Shimadzu Corporation, Kyoto, Japan) system equipped with a RID (RID-20A) and a Sepex SRT SEC-100 column (7.8 × 300 mm, 5 µm). The column was maintained at 35 • C. The 0.7% sodium sulfate solution was used as the mobile phase at a flow rate of 0.5 mL min −1 . A total of 10 µL of sample was injected. The Mw ranges of dextran standards are from 100 Da to 100,000 Da, analyzed by Agilent Chem Station GPC Data Analysis Software (Rev. A.02.01, Agilent Technologies, Palo Alto, CA, USA). The Mw of POPs was calculated and compared with that of dextran standards.

Monosaccharide Composition of POPs
The monosaccharide composition of POPs was determined using the HPLC method of PMP [29]. Briefly, 2 mol L −1 trifluoroacetic acid (2 mL) was added to the polysaccharide sample in a small ampoule, which was then kept in full nitrogen atmosphere for 8 h at 100 • C. The residue was mixed with 1 mL of methanol, then dried by nitrogen stream. The above procedure was repeated five times. The dried residue dissolved in 10 mL of distilled water. The aqueous solution of hydrolyzed polysaccharide or monosaccharide standard mixture was mixed with 5 mL of 0.3 mol L −1 sodium hydroxide. The mixture was treated with a 0.5 mol L −1 methanolic solution (6 mL) of PMP and well mixed. The mixture was incubated at 70 • C for 1 h, then neutralized with 0.3 mol L −1 hydrochloric acid. Extraction was performed three times with an equal volume of chloroform. The aqueous solution was collected and allowed to stand overnight. Following filtration, the aqueous solution was analyzed by HPLC instrument (Agilent1260, Agilent Technologies, Palo Alto, CA, USA) equipped with a Diamonsil C18 analytical column (250 × 4.6 mm, 5 µm). The PMP derivatives were eluted with mobile phases A and B at a flow rate of 0.9 mL min −1 . A total of 10 µL of sample was injected. Mobile phase A contained phosphate buffer (pH 6.8) and acetonitrile in a ratio of 85:15 (v/v, %). Mobile phase B contained PBS (pH 6.8) and acetonitrile in a ratio of 60:40 (v/v, %). The column was maintained at 35 • C, and UV absorbance was set up at 250 nm. A linear gradient was used as follows: 0-10 min, 8% A/B; 10-40 min, 8-13% A/B; 40-51 min, 13-20% A/B; 51-52 min, 0% A/B; and 52-60 min, 0% B. The monosaccharide composition of POPs was compared with standards.

Statistical Analysis
Experiments in this study were performed in triplicate. Data are expressed as mean ± standard deviation (SE), and statistical analysis was performed using Excel and Origin 8.0 software. Means in GraphPad prism 5 were subjected to one-way ANOVA. Means of parameters were analyzed using Student's t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001 were regarded as significant differences.
The results show that 0.4 mM and 0.5 mM H 2 O 2 significantly decreased cell viability compared with the blank group ( Figure 1a) and increased cellular ROS levels ( Figure 1b). H 2 O 2 can cause acute oxidative damage and induce ROS overproduction. In addition, 0.4 mM H 2 O 2 was selected as the final concentration for simulating oxidative stress. The four enzyme-assisted extracted polysaccharides improved cell viability in a dose-dependent manner. Initially, different concentrations of POPs were added to the cells for evaluating the cytotoxicity of POPs using an MTT assay (Figure 1c). Cytotoxicity screening showed that 14 fractions had survival rates above 80% at the indicated concentration. Hence, these concentrations, ranging from 12.5 µg mL −1 to 100 µg mL −1 , were selected for further investigation. VPOP1, VPOP2, β-POP2, and α-POP3 improved cell viability to varying degrees compared with the model group ( Figure 1d) and significantly decreased intracellular ROS levels ( Figure 1e). Among all extracts, VPOP1 showed the strongest protective effect on H 2 O 2 -induced MC3T3 cells.

Apoptotic Morphology of MC3T3 Cells
Hoechst 33342, a blue fluorescent dye, enters normal cell membranes in small amounts, causing them to stain a low blue color. For apoptotic cells, Hoechst 33342 entered apoptotic cells more than normal cells. Hence, the fluorescence intensity in apoptotic cells was stronger than that in normal cells. As shown in Figure 2a, the control group showed an intact nuclear morphology, whereas apoptotic body formation was clearly identified in the H 2 O 2 -treated MC3T3 cells. VPOP1 significantly inhibited the formation of apoptotic bodies, and the inhibitory effect was stronger with the increase in the treatment concentration. This indicated that VPOP1 could inhibit the formation of apoptotic bodies by remarkably reducing the total cellular ROS levels.
AO/EB staining was performed to characterize cell morphology. As shown in Figure 2b, fragmented green patches of early apoptotic cells were observed in the control, H 2 O 2treated, and VPOP1 groups. The control group showed a large number of viable cells with green homogeneous nuclei, whereas apoptotic body formation was clearly identified in the H 2 O 2 -treated group. Apoptotic bodies (necrotic cells) appeared homogenously red and showed damaged cytoplasmic membranes. In addition, late apoptotic cells with green and orange or orange particulate matter were observed in the model group and the lowconcentration VPOP1 group. Furthermore, VPOP1 remarkably reduced the formation of apoptotic bodies in a dose-dependent manner.  apoptotic bodies in a dose-dependent manner.
The apoptosis of cells treated with increasing concentrations (25-100 μg mL −1 ) of VPOP1 was detected using flow cytometry to further evaluate the protective effect of VPOP1 on H2O2-induced MC3T3 apoptosis. As shown in Figure 2c, the occurrence of events in the early apoptotic cells (the lower right quadrant R4) and late apoptotic cells (the upper right quadrant R5) markedly decreased with increasing VPOP1 concentration. These results are consistent with the cell staining data presented above.

VPOP1 against Oxidative Stress via the Nrf2/Keap1 Pathway
The nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) signaling pathway is the direct downstream pathway of ROS and regulates the transcription of ARE-dependent genes to maintain cellular redox homeostasis. As shown in Figure 3a, the expression of Nrf2 and Keap1 was inhibited during H2O2-induced oxidative stress. However, the expression levels of these proteins significantly increased after treatment with increasing concentrations (25-100 μg mL −1 ) of VPOP1. Heme oxygenase-1 (HO-1) and NADPH quinine oxidoreductase 1 (NQO1) are key genes in the Nrf2/ARE pathway. The expression levels of NQO1 and HO-1 significantly increased in the H2O2-induced group but dose-dependently decreased in the VPOP1 group.
The Nrf2/Keap1 system is not only present in mammals but also in zebrafish with similar functions. The optimal H2O2 concentration in zebrafish was selected as 3 mM. As shown in Figure 3b, declined expression of Keap1 and elevated expression of HO-1 and NQO1 proteins were found in the H2O2-exposed groups compared with the control. How- Apoptotic body formation was observed using a fluorescence microscope after Hoechst 33342 staining (a) and acridine orange/ethidium bromide (AO/EtBr) staining (b) after 48 h incubation. Arrows point to apoptotic bodies, which decreased with increasing concentration of VPOP1. Flow cytometry analysis of VPOP1 on the nuclear morphology in MC3T3 cells. Cells were stained with propidium iodide and Annexin V−FITC (c). Experiments were performed in triplicate and the data are expressed as the mean ± SE. * p < 0.05 and ** p < 0.01 were considered as significant compared to the control.
The apoptosis of cells treated with increasing concentrations (25-100 µg mL −1 ) of VPOP1 was detected using flow cytometry to further evaluate the protective effect of VPOP1 on H 2 O 2 -induced MC3T3 apoptosis. As shown in Figure 2c, the occurrence of events in the early apoptotic cells (the lower right quadrant R4) and late apoptotic cells (the upper right quadrant R5) markedly decreased with increasing VPOP1 concentration. These results are consistent with the cell staining data presented above.

VPOP1 against Oxidative Stress via the Nrf2/Keap1 Pathway
The nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) signaling pathway is the direct downstream pathway of ROS and regulates the transcription of ARE-dependent genes to maintain cellular redox homeostasis. As shown in Figure 3a, the expression of Nrf2 and Keap1 was inhibited during H 2 O 2induced oxidative stress. However, the expression levels of these proteins significantly increased after treatment with increasing concentrations (25-100 µg mL −1 ) of VPOP1. Heme oxygenase-1 (HO-1) and NADPH quinine oxidoreductase 1 (NQO1) are key genes in the Nrf2/ARE pathway. The expression levels of NQO1 and HO-1 significantly increased in the H 2 O 2 -induced group but dose-dependently decreased in the VPOP1 group.  The "+" indicates that MC3T3 cells or zebrafish embryos were H2O2−induced, while the "-" indicates that the samples were not treated with VPOP1 or H2O2. Experiments were performed in triplicate and the data are expressed as the mean ± SE. * p < 0.05, ** p < 0.01 and *** p < 0.001 indicate that the values were significantly different from those for the control or H2O2−treated group.

Protective Effect of VPOP1 against H2O2-Treated Zebrafish Embryonic (72 hpf) and Larvae (9 dpf)
As shown in Figure 4a,b, high ROS and lipid peroxidation levels were observed in the H2O2-exposed embryos compared with the control. However, the ROS production and lipid peroxidation levels in the larvae significantly decreased following treatment with VPOP1 prior to H2O2 treatment. Treatment of the zebrafish embryos with VPOP1 inhibited ROS production and lipid peroxidation in a concentration-dependent manner. These results indicate that VPOP1 markedly decreased oxidative stress in the H2O2-exposed zebrafish embryos.
Zebrafish embryos of 9 dpf were immersed in a calcein solution and observed under a fluorescence microscope (Nikon, Tokyo, Japan) to test whether VPOP1 and H2O2 could affect the bone structure of zebrafish embryos. The vertebrae of the H2O2-exposed zebrafish embryos were clearly narrower and had lower fluorescence intensity than those The "+" indicates that MC3T3 cells or zebrafish embryos were H 2 O 2 −induced, while the "-" indicates that the samples were not treated with VPOP1 or H 2 O 2 . Experiments were performed in triplicate and the data are expressed as the mean ± SE. * p < 0.05, ** p < 0.01 and *** p < 0.001 indicate that the values were significantly different from those for the control or H 2 O 2 −treated group.
The Nrf2/Keap1 system is not only present in mammals but also in zebrafish with similar functions. The optimal H 2 O 2 concentration in zebrafish was selected as 3 mM. As shown in Figure 3b, declined expression of Keap1 and elevated expression of HO-1 and NQO1 proteins were found in the H 2 O 2 -exposed groups compared with the control. However, the protein expression in the VPOP1 group was consistent with that in the control group. These results show that VPOP1 can resist apoptosis by regulating the Nrf2-Keap1 pathway to exert an anti-osteoporotic effect.

Protective Effect of VPOP1 against H 2 O 2 -Treated Zebrafish Embryonic (72 hpf) and Larvae (9 dpf)
As shown in Figure 4a,b, high ROS and lipid peroxidation levels were observed in the H 2 O 2 -exposed embryos compared with the control. However, the ROS production and lipid peroxidation levels in the larvae significantly decreased following treatment with VPOP1 prior to H 2 O 2 treatment. Treatment of the zebrafish embryos with VPOP1 inhibited ROS production and lipid peroxidation in a concentration-dependent manner. These results indicate that VPOP1 markedly decreased oxidative stress in the H 2 O 2 -exposed zebrafish embryos.
Zebrafish embryos of 9 dpf were immersed in a calcein solution and observed under a fluorescence microscope (Nikon, Tokyo, Japan) to test whether VPOP1 and H 2 O 2 could affect the bone structure of zebrafish embryos. The vertebrae of the H 2 O 2 -exposed zebrafish embryos were clearly narrower and had lower fluorescence intensity than those of the controls (Figure 4c). Fluorescence intensity increased in the VPOP1-treated group compared with the H 2 O 2 -treated group. The results demonstrate that H 2 O 2 exposure inhibited bone growth and development. Moreover, treatment with different concentrations of VPOP1 ameliorated the situation in a concentration-dependent manner.

Discussion
The root cause of OP is the disruption of OB and OC homeostasis. ROS overproduction triggers oxidative stress, which inhibits bone resorption. This process leads to the disruption of homeostasis and bone remodeling. A multitude of studies reported that oxidative stress is associated with the occurrence of OP [30], which may trigger the apoptosis, necrosis, or autophagy of OB cells [31]. H 2 O 2 diffuses across biological membranes and induces the apoptosis of various types of cells [32], which eventually initiates or aggravates the pathogenesis of OP. In the present study, treatment of OB with H 2 O 2 reduced cell viability with subsequent increases in intracellular ROS generation and apoptosis compared with the controls. Increasing studies have shown that antioxidant phytochemicals confer protection against H 2 O 2 -induced OB apoptosis [33]. Moreover, in agreement with previous reports, the cytoprotective capabilities of POPs are primarily due to their anti-oxidative properties [13]. Therefore, the protective effects of POPs inhibiting OB apoptosis by ameliorating oxidative stress were explored in the current study.
The available literature suggests that the master eukaryotic redox-active factor Nrf2 plays an essential role in the cellular defense against inflammatory and oxidative stressinduced OP [34]. The amino-terminal Neh2 domain in the six functional Neh domains of Nrf2 controls the binding of Keap1. Oxidative stress generated during H 2 O 2 conditions may switch on electrophiles and oxidants. Electrophiles can modify the reactive cysteines of Keap1 and lead to Keap1 inactivation and Nrf2 stabilization [35]. However, Nrf2 is released after dissociation from Keap1, and then enters the nucleus and binds to a consensus sequence [36]. In normal organisms, Nrf2 binds to Keap1 and exists in the cytoplasm. Under H 2 O 2 induction, the body produces oxidative stress. The phosphorylation of Nrf2 or modification of the -SH group in Keap1 not only releases Nrf2 from Keap1, but also promotes Nrf2 to enter the nucleus to bind Maf protein. After binding proteins, AREs are activated and promote the transcription of HO-1 and NQO-1 [37]. In addition, in primary cells with high HO-1 expression, the expression of microRNAs and regulated mRNAs changes, which can be associated with apoptosis-induced cell death [38]. This study used MC3T3 cells to evaluate the antioxidant effect of POP on H 2 O 2 -induced cell death. VPOP1 treatment not only increased cell viability but also reduced ROS production in H 2 O 2 -induced MC3T3 cells. Staining and flow cytometry results show that VPOP1 regulated the apoptosis of MC3T3 cells in a dose-dependent manner. These phenomena clearly exhibited their protective effects against oxidative damage. Furthermore, VPOP1 treatment notably enhanced Nrf2 transcriptional activity and thus further promoted Keap1 protein expression, which contributed to the suppression of apoptosis in MC3T3 cells. Interestingly, HO-1 and NQO1 protein expression was significantly upregulated in the H 2 O 2 -induced group compared with the control group. The H 2 O 2 -induced group showed low expression of Nrf2 and Keap1 and high expression of HO-1 and NQO-1, which was consistent with the above result. We conjecture that the antioxidant properties of VPOP1 inhibit the formation of electrophiles and promote the expression of Nrf2 and Keap1. In addition, VPOP1 can reduce the binding of Nrf2 to ARE and the protein expression of HO-1 and NQO1.
In pharmacological studies, zebrafish is routinely used for screening in predictive toxicology [39]. We also tested our hypothesis using zebrafish embryos as an in vivo model system. The mRNA levels of genes for NQO1 and HO-1 increased, whereas Keap1 decreased in the treated groups compared with their controls. The increase in Keap1 protein levels in the VPOP1-treated fish confirmed its key regulatory role in H 2 O 2 -induced oxidative stress. Furthermore, VPOP1 inhibits H 2 O 2 -induced ROS production and lipid peroxidation in zebrafish. Lipid peroxidation is usually triggered by excess ROS, and H 2 O 2 can induce lipid peroxidation in cells [40]. Lipid peroxidation is a chain reaction in which lipids react with free radicals, and lipid hydroperoxides and peroxy fatty acid free radicals generated during this process attack different biomolecules. The attacked biomacromolecules induce corresponding physicochemical changes, and eventually cause damage [41]. VPOP1 showed a strong protective effect on ROS production and lipid peroxidation in H 2 O 2 -treated zebrafish embryos. The larval-stage zebrafish were stained with calcein to confirm whether VPOP1 can alleviate OP caused by oxidative damage. After entering the zebrafish, calcein can bind to the calcified bone matrix in the bones, producing strong green fluorescence. Therefore, calcein can be used to mark zebrafish's osteogenic bone structure, and it is the best stain for living zebrafish bone [42]. The present results show that treatment with different concentrations of VPOP1 ameliorated the H 2 O 2 -exposed inhibited bone growth and development in a concentration-dependent manner. These results suggest that VPOP1 exerts a protective effect against oxidative-stress-induced OP.
In the process of cellulase-assisted polysaccharide extraction, the internal sites of polysaccharide chains are attacked to generate small oligosaccharides with varied lengths. Hence, the entrapped molecules are easily released, thereby increasing the yield [22]. Furthermore, the high antioxidant activity of polysaccharides by viscozyme-assisted extraction can be attributed to the polysaccharide chains containing more galactose sugars and mannose [21]. In our study, the polysaccharides obtained from POL using different enzymes were characterized. Among four enzyme-assisted extractions, cellulase-assisted extraction had the highest yield. The viscozyme-assisted POP extracts exhibited high antioxidant activity. In addition, the conventional method for obtaining polysaccharides from plant species is water extraction and alcohol precipitation, and step-type alcohol precipitation is rarely used in the purification of polysaccharides [43]. Hence, in this study, the polysaccharides of different fractions were extracted by step gradient ethanol precipitation. VPOP1 and VPOP2 with different MW distributions obtained by step gradient ethanol precipitation showed higher activities than the other extracts. VPOP1 was an acidic heteropolysaccharide comprising mannose. The composition of VPOP1 (Mw = 7.6 kDa) confirmed that small-molecule polysaccharide fractions of POL can be developed as natural antioxidants for the treatment of free-radical-related diseases [44]. Experiments using MC3T3 cells and zebrafish embryos demonstrated the antioxidant activity of POP. VPOP1 reduces H 2 O 2 -induced oxidative stress in MC3T3 and zebrafish at concentrations ranging from 25 to 100 µg mL −1 , thereby reducing apoptosis through mitochondria-mediated apoptotic pathways. These effects are carried out by activating the Nrf2/keap1 antioxidant pathway. The results show that POPs exerted protective effects and inhibited OB apoptosis by ameliorating oxidative stress. However, future research should conduct in-depth investigations to elucidate the causal relationship between oxidative stress and OP.

Conclusions
Among four enzyme-assisted extractions, cellulase-assisted extraction had the highest yield. VPOP1 with strong anti-osteoporosis activity was screened as an acidic heteropolysaccharide comprising mannose. VPOP1 protected osteoblasts from oxidative damage in vivo and in vitro by regulating the Nrf2-Keap1 pathway. Hence, low-Mw VPOP1 from POL can be developed as a potential anti-osteoporotic functional food.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data, models, and code generated or used during the study appear in the submitted article.

Conflicts of Interest:
The authors declare no conflict of interest.